Anti-cd137 antibodies and  uses thereof

ABSTRACT

Disclosed herein are agonistic anti-CD137 antibodies and methods of using such for eliciting CD137 signaling, thereby enhancing immune responses such as T cell functions. The antibodies disclosed within may be used to treat diseases, such as cancer and immune disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of International Application No. PCT/CN2017/114569, entitled “Anti-CD137 Antibodies and Uses Thereof,” filed Dec. 5, 2017, the contents of which are herein incorporated by reference in their entirety.

BACKGROUND OF INVENTION

CD137, also known as 4-1BB or tumor necrosis factor receptor subfamily 9 (TNFRSF9), is a member of the tumor necrosis factor (TNF) receptor family. It is expressed by activated T cells (more prevalently by CD8⁺ than CD4⁺), as well as dendritic cells, B cells, follicular dendritic cells, natural killer cells, granulocytes, and in the cells of blood vessel walls at sites of inflammation.

CD137 is a co-stimulator receptor for activated T cells and the crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137 can also induce proliferation in peripheral monocytes, enhance T cell apoptosis induced by TCR/CD3-triggered activation and regulated CD28 co-stimulation, resulting in the promotion of Th1 cell responses. Its expression is induced by lymphocyte activation, and TNF receptor associated factor (TRAF) adaptor proteins, in addition to CD137 ligand (CD137L) have been found to bind to the receptor, leading to the transduction of signals activating NF-κB.

Both antagonistic and agonistic antibodies specific to CD137 have been developed. U.S. Pat. Nos. 6,569,997, 8,137,667, and Fisher et al., Cancer Immunol Immunother (2012) 61:1721-1733. Agonist antibodies specific to CD137 have been reported to enhance T-cell function and promote anti-tumor activity. Fisher et al., Cancer Immunol Immunother (2012) 61:1721-1733. On the other hand, agonist antibodies specific to CD137 have also been reported to induce significant liver toxicity in patients. Segal et al., Clin Can Res., 2017, 23: 1929-1936.

It is therefore of interest to develop effective and safe CD137 agonists for therapeutic applications.

SUMMARY OF INVENTION

The present disclosure is based, at least in part, on the development of agonistic anti-CD137 antibodies that binds CD137 with high affinity and potently enhanced immune cell activity. Such antibodies also showed superior anti-tumor effects. Thus, these agonistic anti-CD137 antibodies, as well as their functional variants, are expected to be effective in treating diseases such as cancer or autoimmune diseases, via, for example modulating immune responses, such as activities of CD4⁺ cells, CD8⁺ cells, dendritic cells, and/or natural killer cells.

Accordingly, aspects of the present disclosure relate to isolated antibodies that bind to CD137 and elicit cell signaling mediated by CD137.

In some embodiments, the anti-CD137 antibodies described herein bind to the same epitope of CD137 as a reference antibody of LYV370 (20A12), LYV371 (11E10), LYV372 (23D2), LYV375 (22F2), LYV390 (30C11), and LYV402 (26B3) or competes against the reference antibody from binding to the epitope. Such an antibody may comprise the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as the reference antibody. In some examples, the isolated antibody comprises the same heavy chain variable region (V_(H)) and the same light chain variable region (V_(L)) as the reference antibody.

In some embodiments, the anti-CD137 antibody described herein binds to human CD137. Such an antibody may cross-react with CD137 from another species (for example, mouse, rat, or a non-human primate).

Any of the anti-CD137 antibodies described herein can be a full-length antibody (e.g., an IgG molecule), which may contain an Fc variant having enhanced binding affinity to FcγRIIB or an antigen-binding fragment thereof (e.g., Fab or a single-chain antibody). In some embodiments, the anti-CD137 antibody described herein is a human antibody or humanized antibody. Alternatively, the antibody may be a chimeric antibody, which may comprise a human heavy chain constant region or a fragment thereof, and/or a human light chain constant region or a fragment thereof. In some examples, the antibody is a bi-specific antibody capable of binding to both CD137 and FcγRIIB.

In another aspect, the present disclosure includes an isolated nucleic acid or a set of nucleic acids, which encode (e.g., collectively) any of the anti-CD137 antibodies described herein. In some embodiments, the nucleic acid or set of nucleic acids are located on one or two vectors, which may be expression vector(s).

The present disclosure, in another aspect, provides a host cell comprising the isolated nucleic acid or set of nucleic acids as described herein. Such a host cell may be used for producing the anti-CD137 antibody. For example, the host cell may be cultured under conditions allowing for expression of the antibody, which can then be harvested from the cell culture (e.g., from the culture medium).

An additional aspect of the disclosure includes a pharmaceutical composition comprising an anti-CD137 antibody as described herein, or a nucleic acid/nucleic acid set encoding such, and a pharmaceutically acceptable carrier.

The present disclosure also provides, in another aspect, a method of modulating immune responses in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, which comprises the anti-CD137 antibody or the encoding nucleic acid(s). In some embodiments, the subject in need thereof is a human patient having, suspected of having, or at risk for a target disease as described herein, for example, cancer or an immune disease such as autoimmune disease. In some examples, the subject (a human patient) to be treated by any of the methods described herein have been undergone or is undergoing a therapy for the target disease, for example cancer or an immune disorder (e.g., an autoimmune disease).

Also within the scope of the present disclosure are pharmaceutical compositions for use in treating cancer or an immune disease such as autoimmune disease, wherein the pharmaceutical compositions comprising any of the anti-CD137 antibodies described herein or encoding nucleic acid(s) thereof, and uses of the antibody or encoding nucleic acid(s) for manufacturing a medicament for use in treating the target disease.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

FIG. 1 is a graph showing dose response of the anti-CD137 antibodies as indicated in binding to human CD137 expressed on CHO cells by FACS analysis.

FIG. 2 is a pair of bar graphs showing the activity of the anti-CD137 antibodies as indicated in a co-stimulation assay with human CD8⁺ T cells from two healthy donors.

FIG. 3 is a sequence alignment of the V_(H) and V_(L) sequences of the anti-CD137 antibodies as indicated. The complementary determining regions (by Kabat CDR definitions) are identified (boxed). The V_(H) sequences, from top to bottom, correspond to SEQ ID NOs: 1, 9, 3, 5, 7, and 11. The V_(L) sequences, from top to bottom, correspond to SEQ ID NOs: 2, 6, 8, 10, 4, and 12.

FIG. 4 is a graph showing the binding of recombinant chimeric antibodies as indicated to human CD137 protein as determined by ELISA.

FIG. 5 is a graph showing the binding of chimeric antibodies as indicated to cellular human CD137 as determined by FACS.

FIG. 6 is a graph showing binding of chimeric antibodies as indicated to cellular cynomolgus monkey CD137 as determined by by FACS.

FIG. 7 includes two graphs showing the activity of chimeric antibodies as indicated in activated human CD8⁺ T cells in vitro. T cells from two donors were tested under conditions where the FcγRIIB-dependent activity of the antibodies was demonstrated.

FIG. 8 is a set of graphs showings pharmacokinetics of chimeric antibodies as indicated in mice.

FIG. 9 is a graph showing the anti-tumor activity of chimeric antibodies as indicated in a tumor model using PC3 cancer cells mixed with human PBMC.

DETAILED DESCRIPTION OF INVENTION

Provided herein are agonistic antibodies capable of binding to CD137 and enhancing the signaling mediated by CD137. Such anti-CD137 antibodies are found to bind to CD137 (e.g., human or monkey) with high affinity and promote T cell activity. The anti-CD137 antibodies described herein were also found to successfully reduce tumor growth in an animal model.

CD137 (also known as 4-1BB or TNFRSF9), is a member of the tumor necrosis factor (TNF) receptor family. It is expressed by activated T cells (more prevalently by CD8⁺ than CD4⁺ (Gramaglia et al., Eur. J. Immunol., 30(2):392-402 (2000)), as well as dendritic cells, B cells, follicular dendritic cells, natural killer cells, granulocytes, and in the cells of blood vessel walls at sites of inflammation. CD137 expression on dendritic cells has been shown to lead to the secretion of IL-6 and IL-12, as well as an increased ability of the DC to stimulate T cell responses to alloantigens as well as to infiltrate tumors (Pan et al., J. Immunol., 172(8):4779-89 (2004)). Activated natural killer cells express CD137 after stimulation with cytokines, promoting the proliferation of natural killer cells and IFN-γ secretion without affecting cytolytic activity (Wilcox et al., J. Immunol., 169(8):4230-6 (2002)).

Agonistic anti-CD137 antibodies may mimic the activity of a natural ligand of CD137 and trigger the cell signaling mediated by CD137 to induce T cell proliferation while preventing activation-associated cell death. Therefore, the agonistic anti-CD137 antibodies can be used to modulate immune responses, for example, increase the activity of T cells and other immune cells, thereby benefiting treatment of diseases such as cancer. It has also been reported that agonistic anti-CD137 antibodies could be effective in treating immune disorders via modulating activities of T cells, which, in turn, regulates activities of other types of immune cells, such as natural killer cells, B cells, macrophages, etc. For example, agonistic anti-CD137 antibodies may benefit treatment of autoimmune diseases via, e.g., preferentially activating CD8⁺ T cells while reducing the activities of CD4⁺ T cells, natural killer cells, and/or B cells, leading to an induction of high IFN-γ levels and modulation of the Th17-Treg cell balance (Vinay et al., Expert Opin on Ther Targets, 2016, 20(3): 361-373; Kim et al., J Immun., 2011, 187: 1120-1128). Accordingly, described herein are anti-CD137 antibodies (e.g., agonistic anti-CD137 antibodies), nucleic acids encoding such, pharmaceutical compositions comprising the antibody or the encoding nucleic acid(s), as well as uses of such antibodies in therapeutic applications.

Antibodies Binding to CD137

The present disclosure provides antibodies that bind CD137, which may be of any source, for example, human and/or monkey CD137. Such antibodies may be agonistic antibodies, which, upon binding to CD137, elicit cell signaling medicated by CD137.

CD137 is a protein well known in the art. For example, NCBI GenBank Accession Nos. NP_001552.2 and NP_001253057.1 provide information for human and cynomolgus monkey CD137, respectively. Provided below are amino acid sequences of exemplary human and cynomolgus monkey CD137 polypeptides.

Human CD137 (SEQ ID NO: 26): MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPP NSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCS MCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNG TKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALL FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL Cynomolgus Monkey (Macaca mulatta) CD137 (SEQ ID NO: 27): MGNSCYNIVATLLLVLNFERTRSLQDLCSNCPAGTFCDNNRSQICSPCPP NSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECS MCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNG TKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQIIFFLALTSTVVLF LLFFLVLRFSVVKRSRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCEL

CD137 polypeptides from other species are known in the art and can be obtained from publicly available gene database, for example, GenBank, using either the human sequence or the cynomolgus monkey sequence as a query.

An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

A typical antibody molecule comprises a heavy chain variable region (V_(H)) and a light chain variable region (V_(L)), which are usually involved in antigen binding. The V_(H) and V_(L) regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each V_(H) and V_(L) is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs.

The anti-CD137 antibody described herein may be a full-length antibody, which contains two heavy chains and two light chains, each including a variable domain and a constant domain. Alternatively, the anti-CD137 antibody can be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, V_(L) and V_(H), are coded for by separate genes, they can be joined using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_(L) and V_(H) regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

In some embodiments, the anti-CD137 antibody as described herein can bind and elicit cell signaling mediated by CD137. The agonistic activity of an anti-CD137 antibody described herein can be determined by routine methods known in the art.

The antibodies described herein can be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries).

Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, variable regions of V_(H) and V_(L) of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human V_(H) and V_(L) chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V_(H) and V_(L) sequences as search queries. Human V_(H) and V_(L) acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region or a part thereof and/or a light constant region or a part thereof from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

In some embodiments, the anti-CD137 antibodies described herein specifically bind to the corresponding target antigen (e.g., CD137) or an epitope thereof. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (CD137) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (i.e., only baseline binding activity can be detected in a conventional method). Alternatively, or in addition, the anti-CD137 antibody described herein may specifically binds human CD137 or a fragment thereof as relative to the monkey counterpart, or vice versa (e.g., having a binding affinity at least 10-fold higher to one antigen than the other as determined in the same assay under the same assay conditions). In other instances, the anti-CD137 antibody described herein may cross-react to human and a non-human CD-137 (e.g., monkey), e.g., the difference in binding affinity to the human and the non-human CD137 is less than 5-fold, e.g., less than 2-fold, or substantially similar.

In some embodiments, an anti-CD137 antibody as described herein has a suitable binding affinity for the target antigen (e.g., CD137) or antigenic epitopes thereof. As used herein, “binding affinity” refers to the apparent association constant or K_(A). The K_(A) is the reciprocal of the dissociation constant (K_(D)). The anti-CD137 antibody described herein may have a binding affinity (K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased K_(D). Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher K_(A) (or a smaller numerical value K_(D)) for binding the first antigen than the K_(A) (or numerical value K_(D)) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. In some embodiments, any of the anti-CD137 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:

[Bound]=[Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A), though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_(A), and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

A number of exemplary anti-CD137 antibodies are provided below (CDRs are in boldface).

20A12D11 V_(H): (SEQ ID NO: 1) QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLET SASTAYLQINNLKNEDMAIYFCARGYGSPDYWGQGTTLTVSS V_(L): (SEQ ID NO: 2) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSGNPPISTFGSGTKLEIKR 11E10D12 V_(H): (SEQ ID NO: 3) QVQLQQSGAELVRPGASVTLSCKASGYTFAGFEMHWIKQTPVHGLGWIGAIDPKTGGTDYNQKFKDKALLTADK SSNTAYMELRSLTSEDSAVYYCTRDLGYFDVWGTGTTVTVSS V_(L): (SEQ ID NO: 4) DIQMTQTTSSLSASLGDRVTISCRASQDIRSNLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDIATYFCQQSEKLPRTFGGGTKLEIRR 23D2D6 V_(H): (SEQ ID NO: 5) QVQLQQSGAELVRPGSSVKISCKASGYAFSIYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFKGKATLTADK SSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSS V_(L): (SEQ ID NO: 6) QIVLTQSPTIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTI SRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKR 22F2C2 V_(H): (SEQ ID NO: 7) EVKLVESGAELVRPGSSVKISCKASGYAFSLYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFKGKATLTADK SSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSS V_(L): (SEQ ID NO: 8) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTI SRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKR 30C11B4 V_(H): (SEQ ID NO: 9) QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMHWVKQAPGKALKWMGLINTYTGKPTYVDDLKGRFVFSLEA SASTAKLQISNLKNEDTAIYFCARYYHDGTYYGWFANWGQGTLVTVSS V_(L): (SEQ ID NO: 10) DTVLTQSPALAVSPGERVTVSCGATESVSTALNWYQQKPGQQPRLLIYGASNLESGVPARFSGSGSGTDFTLSI DPVEADDTATYFCQQTWNDPLTFGSGTKLEIK 26B3D7 V_(H): (SEQ ID NO: 11) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSNPINYTPSLKDKFIISRDN AKNTLYLQMSKVRSEDTALYYCARDGSSSRYFDVWGAGTTVTVSS V_(L): (SEQ ID NO: 12) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGSDFTLS INSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIKR Chimeric antibody: HC of LYV370 = 20A12 in human IgG4SP (SEQ ID NO: 13) QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFS LETSASTAYLQINNLKNEDMAIYFCARGYGSPDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV370 = 20A12 in human kappa (SEQ ID NO: 14) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYS LTISSMEAEDAATYYCQQWSGNPPISTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC Chimeric antibody: HC of LYV371 = 11E10 in human IgG4SP (SEQ ID NO: 15) QVQLQQSGAELVRPGASVTLSCKASGYTFAGFEMHWIKQTPVHGLGWIGAIDPKTGGTDYNQKFKDKALLT ADKSSNTAYMELRSLTSEDSAVYYCTRDLGYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV371 = 11E10 in human Kappa (SEQ ID NO: 16) DIQMTQTTSSLSASLGDRVTISCRASQDIRSNLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQSEKLPRTFGGGTKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C Chimeric antibody: HC of LYV372 = 23D2 in human IgG4S (SEQ ID NO: 17) QVQLQQSGAELVRPGSSVKISCKASGYAFSIYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV372 = 23D2b in human Kappa (SEQ ID NO: 18) QIVLTQSPTIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYS LTISRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Chimeric antibody: HC of LYV375 = 22F2 in human IgG4SP (SEQ ID NO: 19) EVKLVESGAELVRPGSSVKISCKASGYAFSLYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV375 = 22F2 in human Kappa (SEQ ID NO: 20) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYS LTISRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Chimeric antibody: HC of LYV390 = 30C11 in hG4SP (SEQ ID NO: 21) QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMHWVKQAPGKALKWMGLINTYTGKPTYVDDLKGRFVFS LEASASTAKLQISNLKNEDTAIYFCARYYHDGTYYGWFANWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV390 = 30C11 in human kappa (SEQ ID NO: 22) DTVLTQSPALAVSPGERVTVSCGATESVSTALNWYQQKPGQQPRLLIYGASNLESGVPARFSGSGSGTDFT LSIDPVEADDTATYFCQQTWNDPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Chimeric antibody: HC of LYV402 = 26B3b in hG4SP (SEQ ID NO: 23) EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSNPINYTPSLKDKFIIS RDNAKNTLYLQMSKVRSEDTALYYCARDGSSSRYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK Chimeric antibody: LC of LYV402 = 26B3b in human kappa (SEQ ID NO: 24) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGSDF TLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C

In some embodiments, the anti-CD137 antibodies described herein bind to the same epitope of a CD137 polypeptide as any of the exemplary antibodies described herein (for example, LYV370 (20A12), LYV371 (11E10), LYV372 (23D2), LYV375 (22F2), LYV390 (30C11), and LYV402 (26B3)) or compete against the exemplary antibody from binding to the CD137 antigen. An “epitope” refers to the site on a target antigen that is recognized and bound by an antibody. The site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue. An epitope can be linear, which is typically 6-15 amino acids in length. Alternatively, the epitope can be conformational. The epitope to which an antibody binds can be determined by routine technology, for example, the epitope mapping method (see, e.g., descriptions below). An antibody that binds the same epitope as an exemplary antibody described herein may bind to exactly the same epitope or a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residue, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as the exemplary antibody. Whether two antibodies compete against each other from binding to the cognate antigen can be determined by a competition assay, which is well known in the art.

In some examples, the anti-CD137 antibody comprises the same V_(H) and/or V_(L) CDRs as an exemplary antibody described herein. Two antibodies having the same V_(H) and/or V_(L) CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach or the Chothia approach as known in the art). Such anti-CD137 antibodies may have the same V_(H), the same V_(L), or both as compared to an exemplary antibody described herein.

Also within the scope of the present disclosure are functional variants of any of the exemplary anti-CD137 antibodies as disclosed herein. Such functional variants are substantially similar to the exemplary antibody, both structurally and functionally. A functional variant comprises substantially the same V_(H) and V_(L) CDRs as the exemplary antibody. For example, it may comprise only up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in the total CDR regions of the antibody and binds the same epitope of CD137 with substantially similar affinity (e.g., having a K_(D) value in the same order). Alternatively or in addition, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-CD137 antibody may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V_(H) CDRs of an exemplary antibody described herein. Alternatively or in addition, the anti-CD137 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V_(L) CDRs as an exemplary antibody described herein.

The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, the heavy chain of any of the anti-CD137 antibodies as described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can of any suitable origin, e.g., human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from a human IgG (a gamma heavy chain) of any IgG subfamily as described herein. In one example, the constant region is from human Ig molecule such as IgG4, an exemplary amino acid sequence of which is provided below (SEQ ID NO: 25):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCP S CP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK

In some embodiments, the anti-CD137 antibody comprises the heavy chain constant region of SEQ ID NO: 25, or a variant thereof, which may contain an S/P substitution at the position as indicated (boldfaced and underlined). Alternatively, the heavy chain constant region of the antibodies described herein may comprise a single domain (e.g., CH1, CH2, or CH3) or a combination of any of the single domains.

When needed, the anti-CD137 antibody as described herein may comprise a modified constant region. For example, it may comprise a modified constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCC activity can be assessed using methods disclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

Any of the anti-CD137 antibodies described herein may comprise a light chain that further comprises a light chain constant region, which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain.

Antibody heavy and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or at www.vbase2.org/vbstat.php., both of which are incorporated by reference herein.

In some embodiments, the anti-CD137 antibody described herein may contain a mutated Fc region as compared with a wild-type counterpart such that the antibody has a higher binding affinity to an Fc receptor, for example, FcγRIIB (CD32B). Such antibodies may engage FcγRIIB-expressing cells efficiently, thereby enhancing therapeutic effects.

In some embodiments, the anti-CD137 antibody is a bi-specific antibody capable of binding to both CD137 and FcγRIIB (not via Fc-FcR interaction). Such a bi-specific antibody may comprise a first antigen-binding region and a second antigen-binding region, each of which may comprise a V_(H)/V_(L) pair. The first antigen-binding region binds CD137 while the second antigen-binding region binds FcγRIIB

Preparation of Anti-CD137 Antibodies

Antibodies capable of binding CD137 as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

In some embodiments, antibodies specific to a target antigen (e.g., CD137 or a fragment thereof) can be made by the conventional hybridoma technology. The full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the anti-CD137 monoclonal antibodies described herein. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of enhancing CD137 activity. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR, where R and R1 are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to “humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in enhancing the activity of CD137. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Alternatively, antibodies capable of binding to the target antigens as described herein may be isolated from a suitable antibody library via routine practice. Antibody libraries can be used to identify proteins that bind to a target antigen (e.g., CD137) via routine screening processes. In the selection process, the polypeptide component is probed with the target antigen or a fragment thereof and, if the polypeptide component binds to the target, the antibody library member is identified, typically by retention on a support. Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.

There are a number of routine methods known in the art to identify and isolate antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosomal display, or mammalian display technology.

Antigen-binding fragments of an intact antibody (full-length antibody) can be prepared via routine methods. For example, F(ab′)2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.

Techniques developed for the production of “chimeric antibodies” are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, variable regions of V_(H) and V_(L) of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human V_(H) and V_(L) chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V_(H) and V_(L) sequences as search queries. Human V_(H) and V_(L) acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv library and scFv clones specific to CD137 can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that enhance CD137 activity.

Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence, to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of CD137 have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein (such as another member of the tumor necrosis factor receptor family). By assessing binding of the antibody to the mutant CD137, the importance of the particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

In some examples, an anti-CD137 antibody is prepared by recombinant technology as exemplified below.

Nucleic acids encoding the heavy and light chain of an anti-CD137 antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences.

In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.

Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-CD137 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr-CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-CD137 antibody and the other encoding the light chain of the anti-CD137 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.

Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

Any of the nucleic acids encoding the heavy chain, the light chain, or both of an anti-CD137 antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.

Anti-CD137 antibodies thus prepared can be can be characterized using methods known in the art, whereby an increase in CD137 biological activity is detected and/or measured. For example, an ELISA-type assay may be suitable for qualitative or quantitative measurement of CD137 promotion of T cell proliferation.

Methods of Treatment

The present disclosure provides methods of treating a disease, for example a cancer or an immune disorder such as autoimmune disease, by administering a therapeutically effective amount of an anti-CD137 antibody.

Pharmaceutical Compositions

The antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

The antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an antibody with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

Therapeutic Applications

To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as a cancer or an immune disorder such as an autoimmune disease.

Examples of cancers include, but are not limited to, breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

A subject having a target cancer can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. In some embodiments, the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.

Immune disorders refer to a dysfunction of the immune system. Examples include autoimmune diseases, immunodeficiencies, or allergies. In some embodiments, the target disease for treatment is an autoimmune disease. Examples include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis (MG), Graves' Disease, Idiopathic Thrombocytopenia Purpura (ITP), Guillain-Barre Syndrome, autoimmune myocarditis, Membrane Glomerulonephritis, diabetes mellitus, Type I or Type II diabetes, multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis, gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathies, experimental autoimmune encephalomyelitis, immune neutropenia, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines, T-lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus, pemphigold, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, anti-phospholipid syndrome, Sjogren's syndrome, graft-versus-host (GVH) disease, and immune thrombocytopenia.

A subject having a target autoimmune disease can be identified by routine medical examination, e.g., presence of antinuclear antibodies, anti-mitochondrial autoantibodies, anti-neutrophil cytoplasmic antibody, anti-phospholipid antibodies, anti-citrullinated peptide (anti-CCP), anti-rheumatoid factor, immunoglobulin A, C-reactive protein test, complement test, erythrocyte sedimentation rate (ESR) test, blood clotting profile, and protein electrophoresis/immunofixation electrophoresis, among others. In some embodiments, the subject to be treated by the method described herein may be a human subject with an autoimmune disease who has undergone or is subjecting to an autoimmune disease treatment, for example, immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory medication, and/or pain medication.

A subject suspected of having any of such target disease/disorder might show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.

As used herein, “an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is increased CD137 activity, increased T cell proliferation and survival, and/or increased anti-tumor immune responses. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one example, dosages for an antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the agonist. To assess efficacy of the agonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the antibodies described herein, an initial candidate dosage can be about 2 mg/kg. For the purpose of the present disclosure, a typical daily dosage might range from about any of 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing from one-four times a week is contemplated. In some embodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, doses ranging from about 0.3 to 5.00 mg/kg may be administered. In some examples, the dosage of the anti-CD137 antibody described herein can be 10 mg/kg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of an antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agonist, and the discretion of the attending physician. Typically the clinician will administer an antibody, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is an increase in anti-tumor immune response in the tumor microenvironment. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.

As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to enhance the activity of CD137 (and/or T cell proliferation) by at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo.

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.

In one embodiment, an antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisense polynucleotide, expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

Therapeutic compositions containing a polynucleotide (e.g., those encoding the antibodies described herein) are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can also be used during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.

In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of the treatment. The antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed by methods well-known in the art.

Combined Therapy

The anti-CD137 antibodies described herein may be utilized in conjunction with other types of therapy for the target disease such as cancer or immune disorders.

When an anti-CD137 antibody as described herein is used for treating a cancer, it can be combined with an anti-cancer therapy, for example, those known in the art. Additional anti-cancer therapy includes chemotherapy, surgery, radiation, immunotherapy, gene therapy, and so forth.

Alternatively, the treatment of the present disclosure can be combined with a chemotherapeutic agent, for example, pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

When the anti-CD137 antibody as described herein for treating an immune disorder, it can be co-used with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), or checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.). In some instances, the antibody can be combined with another therapy for autoimmune diseases. Examples include, but are not limited to, intravenous Ig therapy; nonsteroidal anti-inflammatory drugs (NSAID); corticosteroids; cyclosporins, rapamycins, ascomycins; cyclophosphamide; azathioprene; methotrexate; brequinar; FTY 720; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine; an immunosuppressive agent, or an adhesion molecule inhibitor.

For examples of additional useful agents see also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

When a second therapeutic agent is used, such an agent can be administered simultaneously or sequentially (in any order) with the therapeutic agent described herein. When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

Kits for Use in Treatment of Diseases

The present disclosure also provides kits for use in treating or alleviating a target diseases, such as cancer and immune disorders as described herein. Such kits can include one or more containers comprising an anti-CD137 antibody, e.g., any of those described herein, and optionally a second therapeutic agent to be co-used with the anti-CD137 antibody, which is also described herein.

In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the anti-CD137 antibody, and optionally the second therapeutic agent, to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying the diagnostic method as described herein. In still other embodiments, the instructions comprise a description of administering an antibody to an individual at risk of the target disease.

The instructions relating to the use of an anti-CD137 antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease, such as cancer or immune disorders (e.g., an autoimmune disease). Instructions may be provided for practicing any of the methods described herein.

The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CD137 antibody as those described herein.

Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES Example 1: Generation of Anti-CD137 Antibodies Reagents and General Methods

Human CD137/4-1BB/TNFRSF9 protein (cat #838-4B, Fc chimera) and human 4-1BB/TNFRSF9 MAb (Clone 145501, mouse IgG2B, cat #MAB838) were purchased from R&D Systems (USA). A stable CHO cell line expressing human or cynomolgus monkey CD137 was developed by transfection with full length human CD137 cDNA (Gene ID: 3604) or cynomolgus monkey CD137 cDNA (Gene ID: 102127961).

A standard ELISA was used to detect anti-CD137 antibodies. Plates were coated with CD137 protein and incubated with anti-CD137 antibodies. The antibody molecules bound to CD137 were detected using secondary antibodies (goat anti-mouse or rat IgG) conjugated with peroxidase.

FACS was performed to detect anti-CD137 antibodies using the CHO-CD137 cell line. After a period of incubation with antibody samples, CHO-CD137 cells were analyzed by commercially available PE- or FITC-labeled secondary antibodies using FACS.

Hybridoma Development and Lead Antibody Identification

Balb/c and SJLmice and Wistar rats were immunized with recombinant human CD137/4-1BB/TNFRSF9 protein (cat #838-4B, Fc chimera) and pcDNA3.1-human CD137 plasmid. The anti-CD137 serum titers of the immunized animals were monitored using an ELISA with human CD137 and human IgG as controls. The positive titers were further confirmed using FACS to screen CHO-human CD137 cells. Three mice and one rat with the highest titers were sacrificed; the harvested spenocytes were isolated and fused with SP2/0 myeloma cells using a standard hybridoma protocol.

Eighty 96-well plates of mouse or rat hybridomas were screened using an ELISA which measured binding to human CD137 protein. The positive samples were then tested by a human Fc counter screen. Next, these positive CD137-specific clones were tested for binding to CHO-human CD137 by FACS and subjected to two rounds of single cell cloning by limited dilutions. Fourteen stable hybridoma clones (2 rat and 12 murine) were obtained.

Selected hybridoma clones were grown for antibody production. These purified antibodies were evaluated for binding to human cellular CD137 by FACS. FIG. 1 shows the binding results of confirmed hybridoma antibodies. These six antibodies were also tested in a co-stimulation assay for human CD8-positive T cells (Fisher et al., Cancer Immunol Immunother, (2012), 61:1721-1733) and the results are shown in FIG. 2, confirming the potential agonist activity of these antibodies.

Preparation of Chimeric Antibodies

The antibody variable domains of the six confirmed hybridomas were sequenced as follows. In brief, total RNA was extracted from hybridoma cells using NucleoZOL (MACHEREY-NAGEL, cat #740404.2), and then was reverse transcribed to cDNA by 5′-RACE with a SMARTer® RACE 5′/3′ Kit (Clontech, cat #634858). Heavy and light chain variable regions were amplified by PCR with specific primers (Novagen, cat #69831-3), and the amplicons purified with NucleoSpin® Gel and a PCR Clean-up Kit (MACHEREY-NAGEL, cat #740609.25). The amplicons were then cloned into a pMD18-T vector (Takara, cat #D101A) using TA cloning. After the clones were transformed in DH5a cells, 15 single clones carrying V_(H) and V_(L) fragments were analyzed to obtain the sequences of antibody variable region. V_(H)/V_(L) sequences were determined using the following rules: the amino acid sequences of insert fragments from several colonies were identical and the FRs and CDRs (Kabat definition) were found in each sequence. The resulting consensus sequence is believed to be the sequence of the antibody produced by the hybridoma, which is shown in FIG. 3 and SEQ ID NOs: 1-12.

The cDNA sequences encoding the anti-CD137 antibody variable domain sequences were synthesized as chimeras to human IgG4 heavy chain constant regions containing the hinge S228P (EU numbering; Kabat numbering 241) stabilizing mutation (Angal et., Mol. Immunol 30:105, 1993) or human kappa light chain constant region. HEK293 and/or CHO transient expression was carried out with plasmids containing the corresponding heavy and light chain sequences. These chimeric antibodies were purified by protein affinity chromatography. The purified antibodies were checked for endotoxin (<5 EU/mg) and monomerization (>95%).

Example 2: Evaluation of Anti-CD137 Chimeric Antibodies K_(D) Measurement of CD137 Antigen Binding

The chimeric antibodies were tested in an antigen binding assay on Octet Red 96 to estimate binding kinetics. Antibodies were loaded onto anti-human Fc (AHC) biosensors. Loaded sensors were dipped into a serial dilution of each antibody (300 nM, 1:3 down, 7 points) in assay buffer (PBS with 0.1% BSA, 0.02% Tween-20 (pH 7.2)). Kinetic constants calculated using a monovalent (1:1) model are shown in Table 1 below.

TABLE 1 Kinetic Constants of Chimeric Antibodies Antibodies K_(D)(M) k_(on)(1/Ms) k_(dis)(1/s) LYV370 (20A12-IgG4) 5.70E−09 1.80E+05 1.00E−03 LYV371 (11E10-IgG4) 3.80E−09 2.80E+05 1.10E−03 LYV372 (23D2-IgG4) 5.80E−09 2.80E+05 1.60E−03 LYV375 (22F2-IgG4) 8.20E−09 2.80E+05 2.30E−03 LYV390 (30C11-IgG4) 5.30E−09 2.60E+05 1.40E−03 LYV402 (26B3-IgG4) 2.10E−09 1.60E+05 3.40E−04

CD137 Binding ELISA

CD137 protein (human CD137-His tag protein (Sino Biological Inc. Cat #10377-H08H-100) or rhesus monkey CD137-His (Sino Biological Inc. Cat #90305-K08H-100)) was diluted in PBS to 1 ug/ml and used to coat an ELISA plate (Corning, Cat #9018, high binding) at a concentration of 50 ul/well. The plate was incubated overnight at 4° C. The plate was then decanted and washed with PBS-T, and 200 ul/well assay diluent (1×PBS/1% BSA/0.05% Tween-20/0.05% proclin 300) was added. After a 3 hour incubation at room temperature, the plate was washed with PBS-T three times. The testing antibodies were diluted in assay diluent to 0, 0.000003, 0.00003, 0.0003, 0.003, 0.03, and 0.3 ug/ml or approximately 0, 0.00002, 0.0002, 0.002, 0.02, 0.2 and 2 nM and then added to the plate (50 ul/well). The plate was incubated for one hour at 37° C. and then washed three times with PBS-T. Anti-human IgG-HRP conjugate (Bethyl Cat #A80-319P) at a 1:10,000 dilution was added to the plate (100 ul/well). The plate was incubated for 0.5 hour at 37° C., followed by washing with PBS-T three times. The TMB substrate solution was added (100 ul/well). The color was allowed to develop for 8 minutes before it was stopped with 100 ul/well 2N H₂SO₄. Absorbance at 450 nm was determined with an ELISA reader. Dose response curves of the chimeric antibodies are shown in FIG. 4.

CD137 Binding FACS

CHO cells over-expressing human CD137 or cynomolgus monkey CD137 were harvested using trypsin-EDTA partial digestion followed by centrifugation at 1000 rpm for 5 minutes. The cells were resuspended in cold PBS-BSA (2%) at 5×10⁶/m1 and aliquoted out to 100 ul/tube. The chimeric anti-CD137 antibodies were diluted in PBS-BSA in three times (final concentrations were 0.01, 0.1, 1, and 10 ug/ml) and 50 ul of each concentration was added to the CHO-CD137 cells. The cell solutions were mixed and incubated at 4° C. in the dark for 2 hours. The cells were then washed with PBS-BSA twice. Secondary antibody conjugates (goat F(ab′)2 anti-human IgG-Fc (PE), pre-adsorbed (ab98596)) at a concentration of 100 ul/vial was added and the cells were mixed and incubated 4° C. in dark for 1 hour. The cells were then washed twice with PBS-BSA, followed by fixation in 2% PFA, and were then subjected to FACS analysis with FACScaliber. As shown in FIG. 5, these antibodies exhibited saturable binding to the CHO cells over-expressing human CD137. However, binding to cellular cynomolgus monkey CD137 of these antibodies differs, as shown in FIG. 6, as only three antibodies (LYV371, LYV390 and LYV402) exhibited high affinity binding to cynomolgus monkey CD137.

T Cell Functional Assays

Fresh PBMCs were isolated from two healthy volunteers and resuspended in PRMI-1640 containing 10% FBS at 1×10⁶/ml. CD8 T cells were isolated from the samples using EasySep™ Human CD8⁺ T Cell Isolation Kit (Stemcell, 17953). The resulting T cells were diluted into concentrations of 5×10⁵/m1 in RPMI 1640 (10% FBS).

Co-stimulation assays of human CD8-positive T cells were performed under three conditions: no co-culture, co-culture with parental CHO cells, and co-culture with CHO cells expressing human FcγRIIB To run co-culture assays, parental CHO cells or CHO cells engineered to express human FcγRIIB were plated in 96-well culture plates at a concentration of 2.5×10⁴ cells/well. The cells were allowed to attach during an overnight incubation period in a cell culture incubator at 37° C. and 5% CO₂. Human CD8 T cells were added at 1×10⁵ cells/well, OKT3 was added at 0.1 ug/mL, and CD137 antibodies were added at 0, 0.03, 0.1, 0.3, 1, and 3 ug/mL final concentration. The culture plates were incubated for 3 days in cell culture incubator at 37° C. and 5% CO₂. The IFNγ content in the culture supernatants was determined by ELISA (eBioscience, 88-7316-88). As shown in FIG. 7, chimeric antibodies showed the ability to co-stimulate human CD8-positive T lymphocytes in the presence of FcγRIIB-expressing CHO cells.

Example 3: Pharmacokinetic Study of Chimeric Antibodies

C57BL/6 mice (6-7 weeks old, 19-20 g, male, purchased from SLAC Laboratory Animal Co. LTD) were used for the study. Antibodies were formulated in PBS and administered via tail vein injection at 3 mg/kg in a group of 4 mice.

Blood sampling was done at pre-dose, 1 h, 2 h, 4 h, 8 h, 1 d, 2 d, 3 d, 5 d, 8 d, 11 d, 15 d and 21 d by serial bleeding. 10 uL blood per time point was added to 40 uL of a PBS-BSA solution. The sample was then mixed well and centrifuged at 2000 g for 5 minutes at 4° C. The supernatant was put on dry ice immediately after collection and stored at approximately −70° C. until analysis. Blood antibody concentrations were determined by ELISA as described in section above. FIG. 8 shows the blood antibody concentration of chimeric antibodies after a single intravenous injection of 3 mg/kg.

Example 4: In Vivo Evaluation of Efficacy of Antibody Treatment of PC-3 Human Prostate Cancer (MiXeno Model)

PC-3 tumor cells were maintained in vitro as a monolayer culture in Ham's F12K medium supplemented with 10% FBS at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

Human PBMCs were isolated from the blood of two healthy donors (donor A and donor B). After centrifugation, the cells were washed with a PBS solution and resuspended in PBS. The cell number was adjusted to 1.5×10⁷ cells/ml (1.5×10⁶/100 ul) for inoculation.

SCID/Beige mice of 8-10 weeks of age with 18.5-21.5 g body weight were used for the study. All mice were sub-lethally irradiated with 60Co (150 rad) one day before tumor inoculation. First, a total of 104 mice were grouped into two cohorts by body weight. Mice in each cohort were inoculated subcutaneously in the right flank region with 3×10⁶ PC-3 tumor cell/mouse premixed with 1.5×10⁶ PBMCs from donor A or B in 0.2 ml PBS. The date of tumor cell inoculation was denoted as day 0. On day 1, mice were assigned to groups (n=5) based on body weight, and dosing began. Before grouping and treatment, all animals were weighed and the tumor volumes were measured using a caliper. Since the tumor volume can affect the effectiveness of any given treatment, tumor volume was used as numeric parameter to randomize selected animals into specified groups. Thus, the systematic error was minimized. The grouping was performed by using StudyDirector™ software (Studylog Systems, Inc. CA, USA). One optimal randomization design (generated by a Matched distribution) showed minimal group to group variation in tumor volume based on the group allocation.

After tumor cell inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss (body weights were measured twice weekly after grouping), eye/hair matting and any other abnormal effects. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. On day 1 and day 5, antibodies at 3 mg/kg or the control vehicle were administered via intraperitoneal injections. Following the grouping, tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V=0.5 a×b² where a and b are the long and short diameters of the tumor, respectively. The entire dosing procedure, as well as the tumor and body weight measurements, was conducted in a Laminar Flow Cabinet. The tumor growth curves are shown in FIG. 9, which indicates that two of the antibodies, LYV371m and LYV372m, showed antitumor activity.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 

1. An isolated and non-naturally occurring anti-CD137 antibody, which binds to the same epitope of CD137 as a reference antibody selected from the group consisting of 20A12D11, 30C11B4, 11E10D12, 23D2D6, 22F2C2, and 26B3D7.
 2. The isolated and non-naturally occurring antibody of claim 1, wherein the anti-CD137 antibody binds to a human CD137.
 3. The isolated and non-natural occurring antibody of claim 1, wherein the anti-CD137 antibody binds to both a human CD137 and a non-human CD137.
 4. The isolated and non-natural occurring antibody of claim 3, wherein the non-human CD137 is a cynomolgus monkey CD137.
 5. The isolated and non-natural occurring antibody of claim 1, wherein the antibody comprises the same heavy chain complementary determining regions (CDRs) and the same light chain CDRs as the reference antibody.
 6. The isolated and non-naturally occurring antibody of claim 5, wherein the antibody comprises the same heavy chain variable region (V_(H)) and the same light chain variable region (V_(L)) as the reference antibody.
 7. The isolated and non-natural occurring antibody of claim 1, wherein the antibody is a full length antibody or an antigen-binding fragment thereof.
 8. The isolated and non-natural occurring antibody of claim 7, wherein the antibody is a full-length antibody which is an IgG molecule.
 9. The isolated and non-natural occurring antibody of claim 7, wherein the antibody is a Fab or single-chain antibody.
 10. The antibody of claim 1, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
 11. The antibody of claim 1, wherein the antibody is a bi-specific antibody that binds both CD137 and FcγRIIB.
 12. The antibody of claim 11, wherein the chimeric antibody comprises a human heavy chain constant region or a fragment thereof, a human light chain constant region or a fragment thereof, or both.
 13. An isolated nucleic acid or set of nucleic acids which collectively encode an anti-CD137 antibody of claim
 1. 14. The isolated nucleic acid or set of nucleic acids of claim 13, wherein the nucleic acid or set of nucleic acids are located on one vector or on two vectors.
 15. The isolated nucleic acid or set of nucleic acids of claim 14, wherein the one or two vectors are one or two expression vectors.
 16. A host cell comprising the isolated nucleic acid or set of nucleic acids of claim
 14. 17. A pharmaceutical composition, comprising an anti-CD137 antibody of claim
 1. 18. A method of modulating immune responses in a subject, the method comprising administering to a subject in need thereof an effective amount of an pharmaceutical composition of claim
 17. 19. The method of claim 18, wherein the subject in need thereof is a human patient having, suspected of having, or at risk for a cancer or an immune disorder.
 20. The method of claim 19, wherein the cancer is selected form the group consisting of prostate cancer, colon cancer, and melanoma.
 21. The method of claim 29, wherein the subject in need thereof is a human patient having, suspected of having, or at risk for an autoimmune disease selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Type I diabetes, multiple sclerosis, Celiac Disease, and graft-versus-host (GVH) disease.
 22. The method of claim 18, wherein the subject has undergone or is undergoing a therapy for the cancer or the immune disorder.
 23. A method for producing an anti-CD137 antibody, comprising: (i) culturing the host cell of claim 16 under conditions allowing for expression of the anti-CD137 antibody; and (ii) harvesting the anti-CD137 antibody thus produced from the cell culture. 